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- PDB-9h76: Bacterial LAT transporter BASC in complex with L-Ala and NB53 -

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Basic information

Entry
Database: PDB / ID: 9h76
TitleBacterial LAT transporter BASC in complex with L-Ala and NB53
Components
  • Nanobody NB53
  • Putative amino acid/polyamine transport protein
KeywordsMEMBRANE PROTEIN / Transporter / nanobody / aminoacid transporter
Function / homology: / L-amino acid transmembrane transporter activity / Amino acid/polyamine transporter I / Amino acid permease / membrane / ALANINE / Putative amino acid/polyamine transport protein
Function and homology information
Biological speciesCarnobacterium sp. AT7 (bacteria)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFort, J. / Palacin, M.
Funding support Spain, 2items
OrganizationGrant numberCountry
Ministerio de Ciencia e Innovacion (MCIN)PID2021- 122802OB-I00 Spain
Generalitat de Catalunya2021 SGR 01281 Spain
CitationJournal: PNAS Nexus / Year: 2025
Title: The conserved lysine residue in transmembrane helix 5 is pivotal for the cytoplasmic gating of the L-amino acid transporters.
Authors: Joana Fort / Adrià Nicolàs-Aragó / Luca Maggi / Maria Martinez-Molledo / Despoina Kapiki / Paula González-Novoa / Patricia Gómez-Gejo / Niels Zijlstra / Susanna Bodoy / Els Pardon / Jan ...Authors: Joana Fort / Adrià Nicolàs-Aragó / Luca Maggi / Maria Martinez-Molledo / Despoina Kapiki / Paula González-Novoa / Patricia Gómez-Gejo / Niels Zijlstra / Susanna Bodoy / Els Pardon / Jan Steyaert / Oscar Llorca / Modesto Orozco / Thorben Cordes / Manuel Palacín /
Abstract: L-Amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these ...L-Amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these transporters is related to cancer. BasC is a bacterial LAT transporter with an APC fold. In this study, to monitor the cytoplasmic motion of BasC, we developed a single-molecule Förster resonance energy transfer assay that can characterize the conformational states of the intracellular gate in solution at room temperature. Based on combined biochemical and biophysical data and molecular dynamics simulations, we propose a model in which the conserved lysine residue in TM5 supports TM1a to explore both open and closed states within the cytoplasmic gate under apo conditions. This equilibrium can be altered by substrates, mutation of conserved lysine 154 in TM5, or a transport-blocking nanobody interacting with TM1a. Overall, these findings provide insights into the transport mechanism of BasC and highlight the significance of the lysine residue in TM5 in the cytoplasmic gating of LATs.
History
DepositionOct 26, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
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Revision 1.1Feb 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative amino acid/polyamine transport protein
B: Nanobody NB53
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,0433
Polymers60,9542
Non-polymers891
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Putative amino acid/polyamine transport protein


Mass: 46696.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Carnobacterium sp. AT7 (bacteria) / Gene: CAT7_03719 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A8UCQ5
#2: Antibody Nanobody NB53


Mass: 14257.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-ALA / ALANINE


Type: L-peptide linking / Mass: 89.093 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7NO2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BASC-NB53 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 45 kDa/nm / Experimental value: NO
Source (natural)Organism: Carnobacterium sp. (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pttQ18
Buffer solutionpH: 7.4
SpecimenConc.: 2.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 0.022 sec. / Electron dose: 1.04 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 38963

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4.4.1particle selectionTemplate Picker
4cryoSPARCv4.4.1CTF correctionPatch Ctf
9cryoSPARCv4.4.1initial Euler assignmentAb-initio
10cryoSPARCv4.4.1final Euler assignmentNU-refinement
11cryoSPARCv4.4.1classification3D classification
12cryoSPARCv4.4.13D reconstructionNU refinement
13PHENIXmodel refinement
Image processingDetails: 32312 images selected after Ctfs and Motion correct.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2000000
Details: particle picked using CS template picker with low resolution volume abtained previously
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324910 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054255
ELECTRON MICROSCOPYf_angle_d0.5925806
ELECTRON MICROSCOPYf_dihedral_angle_d4.364592
ELECTRON MICROSCOPYf_chiral_restr0.042698
ELECTRON MICROSCOPYf_plane_restr0.005710

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